U.S. patent application number 12/196129 was filed with the patent office on 2009-06-18 for method of synthesizing a suppressor trna, dna construct and use thereof for producing a non-natural amino acid-incorporated protein.
This patent application is currently assigned to RIKEN. Invention is credited to Nobumasa Hino, Takatsugu Kobayashi, Takahito Mukai, Kensaku Sakamoto, Shigeyuki YOKOYAMA.
Application Number | 20090155844 12/196129 |
Document ID | / |
Family ID | 38458965 |
Filed Date | 2009-06-18 |
United States Patent
Application |
20090155844 |
Kind Code |
A1 |
YOKOYAMA; Shigeyuki ; et
al. |
June 18, 2009 |
METHOD OF SYNTHESIZING A SUPPRESSOR tRNA, DNA CONSTRUCT AND USE
THEREOF FOR PRODUCING A NON-NATURAL AMINO ACID-INCORPORATED
PROTEIN
Abstract
There are provided a DNA construct comprising
non-eukaryote-derived suppressor tRNA gene containing no internal
promoter functioning in a eukaryotic cell, and a eukaryote-derived
or bacteriophage-derived promoter linked at the 5' end of the tRNA
gene, a method for synthesizing a suppressor tRNA by using the DNA
construct, and a process for producing a non-natural amino
acid-incorporated protein by using the same.
Inventors: |
YOKOYAMA; Shigeyuki;
(Yokohama-shi, JP) ; Sakamoto; Kensaku;
(Yokohama-shi, JP) ; Hino; Nobumasa;
(Yokohama-shi, JP) ; Mukai; Takahito;
(Yokohama-shi, JP) ; Kobayashi; Takatsugu;
(Yokohama-shi, JP) |
Correspondence
Address: |
BIRCH STEWART KOLASCH & BIRCH
PO BOX 747
FALLS CHURCH
VA
22040-0747
US
|
Assignee: |
RIKEN
Wako-shi
JP
|
Family ID: |
38458965 |
Appl. No.: |
12/196129 |
Filed: |
August 21, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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PCT/JP2007/053304 |
Feb 22, 2007 |
|
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12196129 |
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Current U.S.
Class: |
435/69.1 ;
435/325; 536/23.1 |
Current CPC
Class: |
C12P 21/00 20130101;
C12N 15/67 20130101; C12N 15/85 20130101; C12P 21/02 20130101; C12P
19/34 20130101 |
Class at
Publication: |
435/69.1 ;
536/23.1; 435/325 |
International
Class: |
C12P 21/00 20060101
C12P021/00; C07H 21/04 20060101 C07H021/04; C12N 5/10 20060101
C12N005/10 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 22, 2006 |
JP |
2006-045788 |
Claims
1. A DNA construct comprising a non-eukaryote-originating
suppressor tRNA gene containing no internal promoter sequence that
functions in a eukaryotic cell, and a eukaryote-originating
promoter linked to the 5' end of said tRNA gene.
2. The DNA construct of claim 1, wherein said tRNA gene is a
pyrrolysine tRNA gene originating from archaebacteria and/or a
suppressor tyrosine tRNA gene originating from Escherichia
coli.
3. The DNA construct of claim 1, further comprising a transcription
terminator sequence linked to the 3' end of said tRNA gene.
4. The DNA construct of claim 1, wherein said eukaryote-originating
promoter is a nucleotide sequence that induces transcription by RNA
polymerase II or III.
5. The DNA construct of claim 4, wherein said nucleotide sequence
that induces the transcription by the RNA polymerase III is a
promoter of a eukaryotic tRNA gene, or a promoter of a U6 snRNA
gene.
6. The DNA construct of claim 5, wherein said promoter of the
eukaryotic tRNA gene is a nucleotide sequence of human valine
tRNA.
7. The DNA construct of claim 5, wherein said promoter of the U6
snRNA gene is formed of a nucleotide sequence set forth in SEQ ID
NO:3 or a nucleotide sequence that is at least 30%, 50%, 70% 90% or
95% homologous thereto, and induces transcription by RNA polymerase
III in a mammalian cell.
8. The DNA construct of claim 4, wherein said nucleotide sequence
that induces the transcription by the RNA polymerase II is a
promoter of a U1 snRNA gene.
9. A method of synthesizing a suppressor tRNA comprising causing
the DNA construct of claim 1 to undergo transcription in a
eukaryotic cell.
10. A recombinant eukaryotic cell that is transformed or
transfected by the DNA construct of claim 1.
11. A process for producing a non-natural amino acid
incorporated-protein comprising: expressing, in the presence of the
non-natural amino acid in a eukaryotic cell, (a) an aminoacyl-tRNA
synthetase for the non-natural amino acid, (b) a tRNA which is
capable of binding to the non-natural amino acid in the presence of
said aminoacyl-tRNA synthetase, and transcribed by the DNA
construct of claim 1, and (c) a desired protein that has a nonsense
mutation or frame shift mutation at a desired position.
12. The process of claim 11, wherein said non-natural amino acid is
a lysine derivative and/or a tyrosine derivative.
13. The process of claim 12, wherein said lysine derivative is one
selected from the group consisting of pyrrolysine,
N.epsilon.-t-butoxycarbonyl-lysine, N.epsilon.-acetyl-lysine,
N.epsilon.-trimethyl-lysine, and
N.epsilon.-2-methylamino-benzoyl-lysine.
14. The process of claim 12, wherein said tyrosine derivative is
3-iodo-tyrosine, 4-azido-L-phenylalanine, or
4-benzoyl-L-phenylalanine.
15. A DNA construct comprising a non-eukaryote-originating
suppressor tRNA gene containing no internal promoter sequence that
functions in a eukaryotic cell, and a promoter originating from
bacteriophage linked to the 5' end of said tRNA gene.
16. The DNA construct of claim 15, wherein said tRNA gene is a
pyrrolysine tRNA gene originating from archaebacteria and/or a
suppressor tyrosine tRNA gene originating from Escherichia
coli.
17. The DNA construct of claim 15, wherein said promoter
originating from the bacteriophage is formed of a T7 promoter of
the nucleotide sequence set forth in SEQ ID NO.13 or a nucleotide
sequence that is at least 30%, 50%, 70% 90% or 95% homologous
thereto, and induces transcription by T7 RNA polymerase in a
eukaryotic cell.
18. A process for producing a non-natural amino acid-incorporated
protein comprising: preparing a DNA construct comprising a
non-eukaryote-originating suppressor tRNA gene containing no
internal promoter sequence that functions in a eukaryotic cell, and
a bacteriophage-originating promoter operably linked to the 5'
terminal region of said tRNA gene, wherein said suppressor tRNA is
capable of binding to the non-natural amino acid in the presence of
an aminoacyl-tRNA synthetase for the non-natural amino acid; and
expressing, in the presence of the non-natural amino acid in a
eukaryotic cell, a tRNA transcribed from said DNA construct, an
aminoacyl-tRNA synthetase for the non-natural amino acid, and a
desired protein that has a nonsense mutation or frame shift
mutation at a desired position.
19. The process of claim 18, wherein said bacteriophage-originating
promoter is a T7 promoter, T3 promoter, or SP6 promoter.
20. The process of claim 18, wherein said suppressor tRNA is
transcribed by expressing a bacteriophage-originating RNA
polymerase in said eukaryotic cell.
Description
TECHNICAL FIELD
[0001] The present invention relates to a method of synthesizing a
tRNA and a DNA construct therefor, particularly to a method of
synthesizing a suppressor tRNA corresponding to a non-natural amino
acid and a DNA construct therefor, as well as a method of producing
a non-natural amino acid-incorporated protein using the above.
BACKGROUND ART
[0002] A non-natural amino acid-incorporated protein (hereinafter
also referred to as "alloprotein") in which an amino acid residue
at a desired position in a protein is replaced with an amino acid
other than 20 different amino acids involved in normal protein
synthesis (a non-natural amino acid) could offer an effective means
of analyzing the function and structure of a protein. Meanwhile,
lysine derivatives include amino acids, such as acetyl-lysine,
methyl-lysine etc., which are synthesized by post-translational
modification. Such amino acids are well-known particularly as those
involved in regulation of gene expression by histone and also known
as those involved in regulation of transcriptional activation,
regulation of protein-protein interaction, and
suppression/promotion of ubiquitination for many types of proteins.
It is expected that many findings concerning acetylation,
methylation etc. of lysine would be available if those lysine
derivatives could be introduced site-specifically into a
eukaryote.
[0003] A pyrrolysyl tRNA synthetase (Py1RS) is a novel aminoacyl
tRNA synthetase (aaRS) found from a methanogenic archaebacterium
(Methanosarcina). A corresponding tRNA (pyrrolysine tRNA) is a
suppressor tRNA, which has a unique secondary structure such as an
unusually small D loop etc. Recently, it was found that in
Escherichia coli, Py1RS and pyrrolysine tRNA do not interact with
endogenous aaRS and tRNA (orthogonality), and pyrrolysine could be
introduced amber codon-specifically into a protein (Non-Patent
Document 1). Further, it has been reported that a wild-type Py1RS
can bind a non-natural amino acid such as N.epsilon.-Boc-L-lysine
to pyrrolysine tRNA in Escherichia coli (Non-Patent Document
1).
[0004] On the other hand, in a mammalian cell, an enzyme that
phosphorylates a tyrosine residue in a protein (tyrosine kinase)
plays an important role in transducing a signal such as growth
stimulating factor from an extracellular region into a nucleus. The
tyrosine kinases include one capable of phosphorylating a tyrosine
derivative and one incapable of phosphorylating a tyrosine
derivative. For example, it was shown that a Src kinase
phosphorylates an iodotyrosine residue but an EGF receptor cannot
do. Thus, it is useful in examining interaction of a desired
protein with various tyrosine kinases in a cell if an alloprotein,
the desired protein into which a tyrosine derivative is
incorporated, could be synthesized in a mammalian cell. For
example, it is important in analysis of signal transduction
mechanism to examine which tyrosine kinase phosphorylates the
desired protein. Further, these non-natural amino acid-incorporated
proteins could be useful in themselves as material for analysis of
the function and structure of a protein, and be a substance having
a novel bioactivity
[0005] As an expression method of an alloprotein like the above in
an animal cell, there has been developed a method of expressing in
an animal cell (A) a mutant tyrosyl tRNA synthetase (hereinafter
referred to as "mutant TyrRS"), which is a variant of a tyrosyl
tRNA synthetase derived from Escherichia coli and has an increased
specificity to a non-natural tyrosine derivative as compared with
the specificity to a tyrosine, (B) a suppressor tRNA originating
from eubacterium such as bacillus, mycoplasma, and staphylococcus
capable of binding to the above tyrosine derivative in the presence
of the above mutant tyrosyl tRNA synthetase, and (C) a desired
protein gene subjected to nonsense mutation or frame shift mutation
at a desired position; and incorporating the above tyrosine
derivative into a position of nonsense mutation or frame shift
mutation of the above protein (Patent Document 1 and Non-Patent
Document 2).
[0006] Hereupon, it is required that the above suppressor tRNA
originating from the non-eukaryote is transcribed by an RNA
polymerase in a eukaryotic cell. In contrast to one kind of RNA
polymerase in prokaryotic cells, it is known that in eukaryotic
cells, three different kind of RNA polymerases I, II, and III
(poll, polII, and polIII) act sharing the functions. Poll
synthesizes ribosomal RNA, PolII synthesizes mRNA, and PolIII
synthesizes 5SrRNA, tRNA, U6 small nuclear RNA (snRNA) etc.
Therefore, tRNA in a eukaryotic cell is synthesized by
transcription by RNA polymerase III. Genes transcribed by the RNA
polymerase III are classified broadly into three groups according
to characters of their promoter structures, the groups including,
as their representative genes, a 5SrRNA gene (Type I promoter), a
tRNA gene (Type II promoter), and a U6 small nuclear RNA gene (Type
III promoter), respectively. The type II promoter, which
transcribes a tRNA, is an internal promoter made up of two regions
in a tRNA coding sequence, the consensus sequences of which are
known as box A and box B. The consensus sequence of the box A
consists of the positions 8-19: TRGCNNAGYNGG (SED ID NO:1), and the
consensus sequence of the box B consists of the positions 52-62:
GGTTCGANTCC (SED ID NO:2). Accordingly, for example, the suppressor
tyrosine tRNA of Bacillus stearothermophilus, although it
originates from a prokaryote, can be expressed in an animal cell
without any alterations, because of the presence of the box A and
box B in its suppressor tyrosine tRNA coding sequence (refer to
Non-patent Document 3, for example).
[0007] Here, incorporation (taking-in) of an amino acid into the
position of the nonsense mutation in the above protein is referred
to as suppression. Because there are only three different types of
stop codons, it is three types of non-natural amino acids at the
maximum that can be incorporated into one type of protein. In vitro
experiments have developed artificial base pairs in addition to
naturally occurring base pairs (refer to Non-patent Documents 4 and
5), and an RNA containing artificial base pairs as mentioned above
can be transcribed in vitro by using an RNA polymerase of T7
bacteriophage. It is expected that the following could be achieved:
increase in the number of codon types, which are now 4.sup.3, by
using artificial base pairs in codons encoding amino acids, and
introduction of a plurality of non-natural amino acids into one
type of protein by getting the codons that do not encode natural
amino acids to encode non-natural amino acids.
[Patent Document 1] WO2004/039989A1
[Non-Patent Document 1] Blight, S. K. et al., Nature, 431, 333-335
(2004)
[Non-Patent Document 2] Sakamoto, K. et al., Nucleic Acids Research
30, 4692-4699 (2002)
[Non-Patent Document 3] M. Sprinzl et al., Nucleic Acids Research
17, 1-172 (1989)
[Non-Patent Document 4] Hirao, I. et al., Nature Biotechnology 20,
177-182 (2002)
[Non-Patent Document 5] Hirao, I. et al., Nature Methods 3, 729-735
(2006)
DISCLOSURE OF THE INVENTION
Problems to be Solved by the Invention
[0008] In the case of the above-mentioned non-eukaryote-originating
suppressor tRNA being expressed in a eukaryotic cell, however, the
problem is raised that, if the tRNA has sequences significantly
different from the consensus sequences of the eukaryote in place of
box A and box B, the sequences do not function as internal
promoter, achieving only an extremely small amount of synthesis by
transcription, or hardly causing transcription itself, in the
eukaryotic cell. For example, the D loop of a pyrrolysine tRNA
originating from methanogenic archaebacterium, which lacks several
bases and is unusually small, does not function as internal
promoter in a eukaryotic cell. Further, a suppressor tyrosine tRNA
of Escherichia coli has the box B consensus sequence in its
sequence but does not contain the box A consensus sequence.
Introduction of the boxes A and B into those tRNAs results in loss
of their functions as tRNA so that alloproteins with incorporated
lysine derivative or tyrosine derivative cannot be synthesized even
if a pyrrolysine tRNA or an E. coli's suppressor tyrosine tRNA with
the boxes A and B being incorporated was used.
[0009] On the other hand, it is unknown whether or not those
suppressor tRNAs having no internal promoter would function as tRNA
in cases where the suppressor tRNAs are expressed using an external
promoter in a eukaryotic cell. That is, although it is required
that base modification and formation of 3-dimensional structure
etc. after transcription are normally conducted in order that tRNA
functions, it remains unknown what intracellular localization a
tRNA transcribed by external promoter other than type II promoter
would present, and whether it would undergo post-transcriptional
modification or not, and further whether it would present
biological functions or not.
Means to Solve the Problems
[0010] After investigations and considerations, the inventors have
found out that a pyrrolysine tRNA originating from methanogenic
archaebacterium or a suppressor tyrosine tRNA of Escherichia coli
can be efficiently expressed in an animal cell by binding to its 5'
end a promoter sequence of a eukaryotic tRNA nucleotide sequence or
U1 and U6 snRNA gene(s). Further, it has been found out that the
tRNA can be efficiently expressed by binding a
bacteriophage-originating promoter sequence to the 5' end of the
tRNA gene and introducing the promoter together with a RNA
polymerase capable of transcription into an animal cell. The
present invention has been accomplished based on those
findings.
[0011] In accordance with a first aspect of the present invention,
there is provided a DNA construct comprising a
non-eukaryote-originating suppressor tRNA gene containing no
internal promoter sequence that functions in a eukaryotic cell, and
a eukaryote-originating promoter linked to the 5' end of the tRNA
gene. It is preferred that the tRNA gene is a pyrrolysine tRNA gene
originating from archaebacteria and/or a suppressor tyrosine tRNA
gene originating from Escherichia coli, and the DNA construct
further comprises a transcription terminator sequence linked to the
3' end of said tRNA gene. In a further preferable exemplary
embodiment, the eukaryote-originating promoter is a nucleotide
sequence that induces transcription by RNA polymerase II or III.
The nucleotide sequence that induces the transcription by the RNA
polymerase II is preferably a promoter of a U1 snRNA gene, for
example. Also, it is particularly preferred that the nucleotide
sequence that induces the transcription by the RNA polymerase III
is a promoter of a eukaryotic tRNA gene such as a human valine tRNA
nucleotide sequence, or a promoter of a U6 snRNA gene, for
example.
[0012] In accordance with a second aspect of the present invention,
there is provided a method of synthesizing a suppressor tRNA
comprising: causing the DNA construct to undergo transcription in a
eukaryotic cell; and there is provided a recombinant eukaryotic
cell that is transformed or transfected by the DNA construct; and a
method of synthesizing an aminoacyl-tRNA comprising expressing a
tRNA transcribed by the DNA construct, and an aminoacyl-tRNA
synthetase corresponding to said tRNA.
[0013] In accordance with a third aspect of the present invention,
there is provided a process for producing a non-natural amino acid
incorporated-protein comprising: expressing, in the presence of the
non-natural amino acid in a eukaryotic cell, (a) an aminoacy-tRNA
synthetase for the non-natural amino acid, (b) a tRNA which is
capable of binding to the non-natural amino acid in the presence of
the aminoacyl-tRNA synthetase, and transcribed by the DNA
construct, and (c) a desired protein that has a nonsense mutation
or frame shift mutation at a desired position.
[0014] In accordance with a forth aspect of the present invention,
there is provided a DNA construct comprising: a
non-eukaryote-originating suppressor tRNA gene containing no
internal promoter sequence that functions in a eukaryotic cell, and
a promoter originating from bacteriophage linked to the 5' end of
the tRNA gene. It is preferred that the tRNA gene is a pyrrolysine
tRNA gene originating from archaebacteria and/or a suppressor
tyrosine tRNA gene originating from Escherichia coli, and the DNA
construct further comprises a transcription terminator sequence
linked to the 3' end of the tRNA gene. In addition, preferably, the
bacteriophage-originating promoter is, but not restricted to, a T7
promoter, T3 promoter, or SP6 promoter.
[0015] In accordance with a fifth aspect of the present invention,
there are provided a method of synthesizing a suppressor tRNA
comprising: causing a DNA construct to undergo transcription in a
eukaryotic cell, the DNA construct comprising a
non-eukaryote-originating suppressor tRNA gene containing no
internal promoter sequence that functions in a eukaryotic cell, and
a promoter originating from bacteriophage linked to the 5' end of
said tRNA gene; and a recombinant eukaryotic cell being transformed
or transfected by the DNA construct and a gene expressing an RNA
polymerase corresponding to the bacteriophage-originating
promoter.
[0016] In accordance with a sixth aspect of the present invention,
there is provided a process for producing a non-natural amino
acid-incorporated protein comprising: preparing a DNA construct
comprising a non-eukaryote-originating suppressor tRNA gene
containing no internal promoter sequence that functions in a
eukaryotic cell, and a bacteriophage-originating promoter operably
linked to the 5' terminal region of the tRNA gene, wherein the
suppressor tRNA is capable of binding to the non-natural amino acid
in the presence of an aminoacyl-tRNA synthetase for the non-natural
amino acid; and expressing, in the presence of the non-natural
amino acid in a eukaryotic cell, (a) a tRNA transcribed from the
DNA construct, (b) an aminoacyl-tRNA synthetase for the non-natural
amino acid, and (c) a desired protein that has a nonsense mutation
or frame shift mutation at a desired position. Preferably, the tRNA
gene is, but not restricted to, a pyrrolysine tRNA gene originating
from archaebacteria and/or a suppressor tyrosine tRNA gene
originating from Escherichia coli. Further, it is preferable that
the non-natural amino acid is, but not restricted to, a lysine
derivative or a tyrosine derivative.
[0017] In accordance with a seventh aspect of the present
invention, there is provided a process for producing a non-natural
amino acid-incorporated protein comprising: preparing a DNA
construct comprising a non-eukaryote-originating suppressor tRNA
gene containing no internal promoter sequence that functions in a
eukaryotic cell, and a bacteriophage-originating promoter operably
linked to the 5' terminal region of the tRNA gene, wherein the
suppressor tRNA is capable of binding to the non-natural amino acid
in the presence of an aminoacyl-tRNA synthetase for the non-natural
amino acid; and expressing, in the presence of the DNA construct
and the non-natural amino acid in a eukaryotic cell, (a) an RNA
polymerase corresponding to the bacteriophage-originating promoter,
(b) an aminoacyl-tRNA synthetase for the non-natural amino acid,
and (c) a desired protein that has a nonsense mutation or frame
shift mutation at a desired position. Preferably, the tRNA gene is,
but not restricted to, a pyrrolysine tRNA gene originating from
archaebacteria and/or a suppressor tyrosine tRNA gene originating
from Escherichia coli. Further, it is preferable that the
non-natural amino acid is, but not restricted to, a lysine
derivative or a tyrosine derivative.
MERITORIOUS EFFECTS OF THE INVENTION
[0018] Using a process in the present invention, it allows a
non-eukaryote-originating tRNA, an aminoacyl-tRNA, to be
efficiently expressed in a eukaryotic cell, and a
non-eukaryote-originating suppressor tRNA containing no internal
promoter sequences (box A, box B) that function in a eukaryotic
cell to be expressed in a eukaryotic cell. It is expected that
using process of the present invention, it allows expression of
tRNA containing an artificial non-natural base in a eukaryotic
cell, and synthesis of an alloprotein containing 4 or more
different types of non-natural amino acids.
[0019] Further, using a process in the present invention, it allows
synthesis of an alloprotein into which there is incorporated a
lysine derivative such as particularly in eukaryotes presented
NE-acetyl-lysine, N.epsilon.-trimethyl-lysine,
N.epsilon.-t-butoxycarbonyl-lysine, fluorescent group-containing
N.epsilon.-2-methylamino-benzoyl-lysine etc., by using a wild-type
aminoacyl-tRNA synthetase originating from archaebacteria.
BRIEF DESCRIPTION OF THE DRAWINGS
[0020] FIG. 1 shows a cloverleaf structure of a pyrrolysine
tRNA;
[0021] FIG. 2 shows a result detected of suppression of Grb2
(111amb) by western blot in Example 1;
[0022] FIG. 3 shows a result detected of suppression of Grb2
(111amb) by western blot in Example 2;
[0023] FIG. 4 shows a result of lacZ amber suppression in the case
of tRNA.sup.Py1 being expressed by using 3 different promoters or
enhancers;
[0024] FIG. 5 shows a result of lacZ amber suppression by
tRNA.sup.Tyr linked to U6 promoter in cases where 3 different
non-natural amino acids were added;
[0025] FIG. 6 shows data of mass spectrum demonstrating that in
Escherichia coli, N.epsilon.-Boc-lysine was incorporated into a
peptide in the presence of Py1RS, pyrrolysine tRNA;
[0026] FIG. 7 shows a result detected of suppression of lacZ (91
amber) in the case of tRNA.sup.Py1 being expressed by using T7
promoter in Example 5. It is apparent therefrom that in the case of
T7RNA polymerase being expressed (T7RNAP+), .beta.-galactosidase
activity detected is significantly high as compared with the case
of T7RNA polymerase being not expressed (T7RNAP-), and the amber
codon of a lacZ gene is suppressed;
[0027] FIG. 8 shows a result detected of suppression of lacZ (91
amber) in the case of tRNA.sup.Tyr being expressed by using T7
promoter in Example 5. It is apparent therefrom that in the case of
T7RNA polymerase being expressed (T7RNAP+), .beta.-galactosidase
activity detected is significantly high as compared with the case
of T7RNA polymerase being not expressed (T7RNAP-), and the amber
codon of a lacZ gene is suppressed; and
[0028] FIG. 9 shows a result detected of suppression of lacZ (91
amber) by cellular staining in the case of tRNA.sup.Ty1 being
expressed by using U1snRNA transcription promoter in Example 6.
PREFERRED MODES FOR CARRYING OUT THE INVENTION
Non-Natural Amino Acid
[0029] Non-natural (Non-naturally occurring) amino acid as may be
used herein includes, for example, lysine derivative or tyrosine
derivative. Lysine derivative, a non-natural amino acid, is
preferably ones, the hydrogen atom bonded to the nitrogen atom at
.epsilon. position of which is replaced with another atom or atomic
group. Lysine derivative includes pyrrolysine,
N.epsilon.-t-butoxycarbonyl-lysine (N.epsilon.-Boc-lysine),
N.epsilon.-acetyl-lysine, N.epsilon.-trimethyl-lysine, and
N.epsilon.-2-methylamino-benzoyl-lysine (Nma-lysine), for example.
Site-specific incorporation of methyllysine or acetyllysine, which
is a modified lysine presented in a eukaryote, into a protein could
produce many findings regarding methylation or acetylation of
lysine. Such alloprotein with the lysine derivative incorporated is
useful as material for analysis of function and structure of the
protein, and could offer a target for drug development. Tyrosine
derivative includes 3- or 4-substituted tyrosine made up of a
tyrosine having a substituent at 3- or 4-position of a phenyl group
thereof. 3-Substituted tyrosine includes 3-halogenated tyrosine
such as 3-iodotyrosine and 3-bromotyrosine. 4-Substituted tyrosine
includes 4-acetyl-L-phenylalanine, 4-benzoyl-L-phenylalanine,
4-azido-L-phenylalanine, O-methyl-L-tyrosine,
4-iodo-L-phenylalanine etc. Those amino acids can be prepared by
known methods and are commercially available.
(Aminoacyl-tRNA Synthetase)
[0030] Aminoacyl-tRNA synthetase as used herein is tRNA synthetase
capable of recognizing a non-natural amino acid and specifically
recognizing a suppressor tRNA to produce a suppressor tRNA
connected to such non-natural amino acid.
[0031] In a preferred exemplary embodiment, Py1RS originating from
methanogenic archaebacterium is provided that is able to recognize
a lysine derivative as amino acid and specifically recognize a
pyrrolysine tRNA (SEQ ID NO:4) used as tRNA in combination to
produce a suppressor tRNA connected by such lysine derivative.
Methanogenic archaebacterium is preferably Methanosarcina mazei (M.
mazei). Py1RS is expressed in a eukaryotic cell, preferably in an
animal cell, particularly preferably in a mammalian cell. In order
to express Py1RS in a cell, for example, a plasmid may be
introduced into the mammalian cell which plasmid is constructed
such that a DNA sequence made up of a Methanosarcina
mazei-originating wild-type gene, added with FLAG tag etc. at its N
terminal region, is amplified using PCR, followed by incorporation
of the resultant DNA sequence into NheI-BamHI site of commercially
available pcDNA3.1 (Invitrogen), pAGE107 (Cytotechnology, 3, 133
(1990)), pAGE103 [J. Biochem. 101, 1307 (1987)] etc.
[0032] In other exemplary embodiments, there can be used various
variants of Escherichia coli-originating TyrRS capable of
specifically recognizing a tyrosine derivative to produce a
suppressor tRNA (SEQ ID NO:5) connected with the tyrosine
derivative. For example, the Escherichia coli-originating TyrRS
variant (V37C195) specifically recognizes 3-iodotyrosine.
Alternatively, it has been reported that a TyrRS variant made up of
Escherichia coli-originating TyrRS with introduced mutation of 5
amino acids at positions 37, 126, 182, 185 and 186 recognized
non-natural amino acid such as 4-azido-L-phenylalanine and
4-benzoyl-L-phenylalanine etc. (Chin, J. W. Et al., Science, 301,
964-967, 2003). Escherichia coli-originating TyrRS (wild-type) does
not react with tRNA.sup.Tyr of eukaryotes, and
prokaryote-originating tRNA.sup.Tyr does not react with TyrRS of
eukaryotes.
(tRNA)
[0033] It is required for tRNA used in combination with the above
aminoacyl-tRNA synthetase to satisfy the requirement that it is
assigned to a nonsense codon other than codons assigned to usual 20
different amino acids, and recognized only by the above non-natural
amino acid-specific aminoacyl-tRNA synthetase but not recognized by
an aminoacyl-tRNA synthetase normally present in a host cell
(orthogonal tRNA); and to be expressed in a eukaryotic cell. In a
case where the aminoacyl-tRNA synthetase is Py1RS, the
corresponding pyrrolysine tRNA is a non-eukaryotic cell-originating
tRNA that has an anti-codon complementary to a nonsense codon and a
3-dimensional structure for functioning as suppressor tRNA, and is
expressed in a eukaryotic cell. That is, in this case, the tRNA is
a suppressor tRNA that satisfies the requirement that it is
assigned to a nonsense codon other than codons assigned to usual 20
different amino acids, and recognized only by the above lysine
derivative-specific Py1RS but not recognized by an aaRS normally
present in a host cell (orthogonality); and is expressed in an
animal cell.
[0034] Here, nonsense codon includes UAG (amber), UAA (ochre), UGA
(opal) etc., but UAG (amber) is preferably used. Instead of
nonsense codon, codon made up of 4 or more bases (preferably 4 or 5
bases) (hereinafter referred to as "frame shift codon") may be
used.
[0035] As mentioned above, expression of tRNA in a eukaryotic cell
requires two internal promoters in a tRNA coding sequence, the
consensus sequence of which is known as box A and box B. FIG. 1
shows a cloverleaf structure of a pyrrolysine tRNA. In FIG. 1, the
mark .largecircle. in a loop at the left (D loop) indicates lack of
a base. As shown in FIG. 1, the pyrrolysine tRNA lacks 3 bases in
the D loop and is extraordinarily small as compared with the D
loops of the other tRNAs. In order to express the pyrrolysine tRNA
in an animal cell, the box A and B sequences were incorporated into
the pyrrolysine tRNA, but it resulted in a drastic change in the
structure of tRNA because of the anomalously small size of the D
loop, and in a failure in retaining its suppressor activity.
(Synthesis of tRNA, Amino Acyl-tRNA)
[0036] In a method of synthesizing aminoacyl-tRNA according to the
present invention, a non-eukaryote-originating tRNA containing no
internal promoter sequence that functions in a eukaryotic cell, a
eukaryote-originating promoter being linked to the 5' end of the
tRNA is caused to undergo transcription in a eukaryotic cell,
preferably in an animal cell, particularly preferably in a
mammalian cell, each of which contains an aminoacyl-tRNA
synthetase. In this case, it is preferable that a transcription
terminator sequence is linked to the 3' end of the tRNA. To be more
specific, an aminoacyl-tRNA of the present invention is obtained in
the following manner: the sequence of a Methanosarcina
mazei-originating wild-type pyrrolysine tRNA was synthesized from
DNA primers, the 5' end of which a eukaryote-originating promoter
is linked to and the 3' end of which a transcription terminator
sequence is linked to, to be incorporated into, for example,
pcDNA3.1 or pCR4Blunt-TOPO (both available from Invitrogen), and
the resulting plasmid is introduced into an animal cell to express
the tRNA, followed by transcription and processing in the animal
cell.
[0037] As the above eukaryote-derived promoter, there can be used a
nucleotide sequence that induces transcription by an RNA polymerase
II or III. The nucleotide sequence that induces transcription by an
RNA polymerase II is preferably a U1snRNA gene promoter. However,
it has been reported that a U6snRNA gene promoter with mutated TATA
box region acts as promoter of the U1snRNA gene promoter-type and
thus such promoter may be used. The nucleotide sequence that
induces transcription by an RNA polymerase III promoter is
preferably a eukaryote-originating tRNA gene or U6snRNA gene
promoter. In this case, it is preferred that the
eukaryote-originating tRNA gene is linked via a linker to the 5'
end of a wild-type pyrrolysine tRNA gene. The linker includes, but
not restricted to, a linker cleaved by BglII, XbaI, XhoI etc. The
tRNA gene linked to the 5' end is one originating from a eukaryote,
which includes, but not restricted to, an animal, a plant, an
insect etc. Thereamong, human-originating tRNA gene is preferable.
An amino acid to which the tRNA should be linked may be any one of
usual 20 different naturally-occurring amino acids, preferably
valine among them.
[0038] Human U6 small nuclear RNA (snRNA) is an RNA species which
is abundantly present in a spliceosome that is formed at the stage
of splicing by pre-mRNA and reaches 4-5.times.10.sup.5 copies per
cell. Its U6 promoter is believed to drive transcription of a small
heterologous RNA, the activity of the transcription being higher
than the activity of transcription using a tRNA promoter. Although
the U6 promoter and the tRNA promoter are both transcribed by
PolII, both are different from each other in location: i.e., the U6
promoter is located at 5' upstream of the structural gene whereas
the tRNA promoter is located in the interior of its own structural
gene. Human U6snRNA promoter has distinctive promoter elements
known as enhancer region (or distal promoter region) and core
region (or proximal promoter region), and is preferably a
nucleotide sequence, which is formed of the nucleotide sequence set
forth in SEQ ID NO:3 or a nucleotide sequence being at least 30%,
50%, 70%, 80%, 90% or 95% homologous to the nucleotide sequence set
forth in SEQ ID NO:3, and which has the activity of transcription
by an RNA polymerase III in a mammalian cell. The degree of
homology between nucleotide sequences can be represented by
percentage of identity of two appropriately aligned nucleotide
sequences, which means incidence of accurately identical amino
acids between the sequences. Appropriate alignment between
sequences for identity comparison may be determined using, for
example, BLAST algorithm (Altschul SF J Mol Biol 1990 Oct. 5;
215(3):403-10).
[0039] In the present invention, further, the above tRNA can be
efficiently transcribed in a eukaryotic cell also by using a
bacteriophage-originating promoter. To be specific, possible
promoter includes, but not restricted to, Escherichia
coli-originating T7 promoter, T3 promoter and SP6 promoter. These
promoters may be inserted into any positions within the 5' terminal
region of the above tRNA gene, but the insertion position is
preferably 10-50 bp upstream from the transcription initiation site
of the gene. In the case of bacteriophage-originating promoter
being used, it is required to use a eukaryotic cell in which a
bacteriophage-originating RNA polymerase corresponding to such
promoter is expressed. To be specific, T7 RNA polymerase, T3 RNA
polymerase, and SP6 RNA polymerase may be used as the above
promoter, which is not restricted thereto. It has been reported
that when expressed in a mammalian cell, T7 RNA polymerase caused
transcription of RNA from DNA containing a T7 promoter sequence,
and the amount of the transcribed RNA reached a maximum of 20% of
the total RNAs in the cell. It has been reported that in order to
prepare a large amount of RNAs as in the case of a T7 RNA
polymerase, a T3 RNA polymerase and a SP6 RNA polymerase were used,
the promoters thereof being as short as 20 bp or less like the T7
promoter, and the RNA polymerases each having substantially the
same ability of RNA transcription (as the T7 RNA polymerase) in a
mammalian cell. As the promoter of bacteriophage-originating RNA
polymerase, there is preferred the T7 promoter of the nucleotide
sequence set forth in SEQ ID NO:13, or a sequence formed of a
nucleotide sequence being at least 70%, 80%, 90% or 95% homologous
to the nucleotide sequence set forth in SEQ ID NO:13 and being
capable of inducing transcription by a T7 RNA polymerase in a
mammalian cell.
(Protein into which a Non-Natural Amino Acid is Incorporated)
[0040] In the present invention, proteins into which a non-natural
amino acid is incorporated are not restricted to particular types.
Such proteins may be any proteins capable of expression and further
be heterologous recombinant proteins. Types of the proteins
include, for example, so-called signaling related proteins,
receptors, growth factors, cell cycle related factors,
transcription factors, translation factors, transport related
proteins, secretory proteins, cytoskeletal proteins, enzymes,
chaperones, or disease related proteins, where the diseases include
cancers, diabetes or genetic disease etc.
[0041] In the present invention, it is required to introduce a
nonsense codon (an amber codon in the case of a suppressor tRNA
being an amber suppressor) or a frame shift codon into a site into
which a non-natural amino acid, in particular a lysine derivative
or a tyrosine derivative, is to be incorporated, whereby a
non-natural amino acid, in particular a lysine derivative, can be
specifically incorporated into the nonsense codon (amber codon)
site or the frame shift codon site. As used herein, it is referred
to as "frame shift mutation" that frame shift in an amino acid
sequence to be translated is caused by deletion or insertion of 1,
2, or 4 bases, and aberrant codon formed at the mutated site is
referred to as "frame shift codon". Preferably, frame shift codon
is codon formed of 4 or 5 bases. It has been tried to extend
genetic code by using 4-base codon in various host cells. In the
case of Escherichia coli, for example, 4-base codon of AGGA is used
as alternative codon that is usable without causing much
disturbance of cell function (Anderson, J. C. et al., Proc. Natl.
Acad. Sci., USA 101, 7566-7571).
[0042] Methods for performing site-specific mutagenesis of a
protein may be any well-known methods, and are not restricted to a
particular one. For example, such mutagenesis may be conducted as
required according to a method described in Gene 152, 271-275
(1995); Methods Enzymol. 100, 468-500 (1983); Nucleic Acids Res.
12, 9441-9456 (1984); Proc. Natl. Acad. Sci. USA 82, 488-492 (1985)
or "Saiboukougaku bessatsu `Sinsaiboukougakujikken protocol`,
Shyujyunshya, 241-248 (1993)", or a method using "QuickChange
Site-Directed Mutagenesis Kit"
(Stratagene)
[0043] In the present invention, expression can be performed in an
animal cell, and thus a non-natural amino acid can be incorporated
into such protein that, in Escherichia coli or a cell-free protein
system, is not or less expressed, or cannot undergo
post-translational modification necessary for changing to an active
form. Various types of such proteins are known to a person of
ordinary skill in the art. For example, there may be synthesized an
alloprotein of, but not restricted to, a tyrosine kinase type
receptor such as human EGFR etc. (Cell, 110, 775-787 (2002)), human
Groucho/TLE1 protein (Structure 10, 751-761(2002)), rat
muscle-specific kinase (Structure 10, 1187-1196 (2002)).
[0044] In the method of the present invention, an alloprotein is
expressed in an animal cell so that a non-natural amino acid, in
particular a lysine derivative, can be incorporated into a
carbohydrate chain-linked glycoprotein. Particularly, in the case
of a type of glycoprotein whose pattern of addition of carbohydrate
chain in a cell-free protein system is different from its original
(natural) pattern, a system in an animal cell of the present
invention is thought to be an effective measure to obtain an
alloprotein to which is added a glycoprotein of a pattern of
interest (an original pattern).
[0045] A protein for incorporation of a non-natural amino acid, in
particular a lysine derivative, may be expressed, for example, as
follows: a gene having a sequence constructed such that its codon
corresponding to the position of a desired amino acid of a desired
protein is replaced with a nonsense codon or a frame shift codon
and a desired tag is added to the C terminus thereof is integrated
into BamHI-XhoI site of pcDNA4/TO etc. to produce a plasmid, which
is introduced into an animal cell, resulting in expression
thereof.
(Host)
[0046] An animal cell as host (cell) used in the present invention
is preferably a mammalian cell in which gene recombination system
is established. Examples of useful mammalian host cell system
include a Chinese hamster ovary (CHO) cell and a COS cell. More
unique examples include SV40-transformed simian kidney CV1 system
(COS-7, ATCC CRL 1651); human embryo kidney system (293 cell or
subcloned 293 cell for growth in suspension culture, J. Gen Virol.,
36:59 (1977)); Chinese hamster ovary cell/-DHFR (CHO, Proc. Natl.
Acad. Sci. USA, 77:4216 (1980)); mouse Sertoli's cell (TM4, Biol.
Reprod., 23:243-251(1980)); human lung cell (W138, ATCC CCL 75);
human liver cell (Hep G2, HB 8065); and mouse breast cancer (MMT
060562, ATCC CCL51). The expression system for each of those host
cells is established, and it is within the technical skill of a
person of ordinary skill in the art to select an appropriate host
cell (from among them).
[0047] Method for introducing a vector into the above host cell
includes, for example, electroporation (Nucleic, Acids Res. 15,
1311-1326 (1987)), calcium phosphate method (Mol. Cell. Biol. 7,
2745-2752 (1987)), lipofection method (Cell 7, 1025-1037 (1994);
Lamb, Nature Genetics 5, 22-30 (1993)), etc. These methods may be
conducted, for example, in accordance with a method described in
Molecular Cloning 3.sup.rd edition, Cold Spring Harbor Laboratory
Press (2001) etc. In accordance with one exemplary embodiment of
the present invention, there is provided a recombinant eukaryotic
cell, preferably a recombinant mammalian cell, transformed or
transfected with an expression vector of the above
non-eukaryote-originating suppressor tRNA.
(Method for Producing a Protein with Incorporated Non-Natural Amino
Acid)
[0048] As an example, expression of an alloprotein with
incorporated lysine derivative is explained below. An animal cell
is incubated under appropriate conditions in a medium (culture)
suitable for the growth of the animal cell (for example, Opti-MEMI
(Gibco BRL) etc. in the case of a CHO cell), the animal cell
containing (A) an expression vector expressing an aminoacyl-tRNA
synthetase, in particular a Py1RS in the animal cell; (B) an
expression vector expressing in the animal cell a Methanosarcina
mazei-originating pyrrolysine tRNA capable of binding to a
non-natural amino acid, in particular a lysine derivative, in the
presence of the above aminoacyl-tRNA synthetase, in particular
Py1RS; (C) an expression vector expressing a desired protein
subjected to nonsense mutation or frame shift mutation at a desired
position; and a non-natural amino acid, in particular a lysine
derivative. In the case of a CHO cell, for example, the cell is
incubated at ca. 37 degrees Celsius for ca. 24 hours.
[0049] Alternatively, in the case of the above pyrrolysine tRNA
expressed by using a bacteriophage-originating promoter, it is
preferable to introduce, in addition to the above (A) to (C), (D) a
vector expressing in an animal cell an RNA polymerase gene capable
of transcription of the above bacteriophage-derived promoter. For
example, as RNA polymerases for transcription of the above T7
promoter, T3 promoter, and SP6 promoter, there are known a T7 RNA
polymerase, a T3 RNA polymerase, and SP6 RNA polymerase,
respectively.
[0050] Some examples of the present invention are detailed below
but it should not be understood that the present invention is
restricted to the examples as mentioned below.
EXAMPLES
[0051] In these examples, there were conducted experiments for
incorporation of a lysine derivative or a tyrosine derivative into
111 position of human Grb2 and 91 position of .beta.-galactosidase.
In this regard, the Grb2 is a protein involved in canceration by
interaction with an epidermal growth factor receptor in a cell.
(Construction of Py1RS and TyrRS Expression Plasmids)
[0052] Py1RS expression plasmid was constructed as follow: a DNA
sequence (SEQ ID NO:8) made up of a Methanosarcina
mazei-originating wild type Py1RS gene, the N terminus region of
which a FLAG tag was linked to, was amplified by PCR, followed by
incorporation of the DNA sequence into NheI-BamHI site of pcDNA3.1
to generate the plasmid.
[0053] On the other hand, there has been reported the expression
plasmid pEYSM1 of 3-iodo-L-tyrosine specific mutant (TyrRS
(V37C195)) of Escherichia coli tyrosyl tRNA synthetase (supra.,
Non-Patent Document 2). Transfection into a mammalian culture cell,
of this plasmid together with an expression plasmid of a suppressor
tRNA followed by addition of 3-iodo-L-tyrosine to a cell culture
solution allows incorporation of the 3-iodo-L-tylosine into the
amber codon site of a protein gene with amber mutation. Method of
preparing the above expression plasmid is described in the above
Patent Document 1 and Non-Patent Document 2, the contents of both
documents being incorporated herein by reference. In addition,
there has been reported mutant(s) specific to
4-azido-L-phenylalanine and 4-benzoyl-L-phenylalanine (Chin et al.,
supra). Those mutants (variants) TyrRS were cloned into multiple
cloning site of pcDNA4/TO.
(Construction of Suppressor tRNA Expression Plasmid)
[0054] A sequence (SEQ ID NO: 11) was synthesized from a DNA
primer. This sequence was made up of Methanosarcina mazei-derived
wild type pyrrolysine tRNA gene, the 5' end of which a human valine
tRNA gene was linked to via a linker (SEQ ID NO:10), further the 5'
and 3' ends of which a leader sequence and a transcription
termination sequence, respectively, were linked to. This sequence
was introduced into pCR4Blunt-TOPO, resulting in construction of a
tRNA.sup.VAL-tRNA.sup.Py1 tandem expression plasmid. For
Escherichia coli-originating suppressor tRNA.sup.Tyr, a
tRNA.sup.VAL-tRNA.sup.Tyr tandem expression plasmid was constructed
in the similar manner.
[0055] A tRNA expression plasmid with a U6 promoter was constructed
in the following method. With a pcDNA3.1 vector being as template,
PCR was performed using a primer made up of a CMV enhancer region
the 5' side of which EcoRI site was added to and the 3' side of
which a portion of the 5' side sequence of a U6 promoter was added
to. And with an siSTRIKE being as template, PCR was performed using
a primer made up of a U6 promoter region at the 5' side of which
there was contained a portion of the 3' side sequence of a CMV
enhancer and to the 3' side of which XbaI site was added. The two
different PCR amplification fragments were joined to each other by
overlap PCR to produce a DNA fragment formed of EcoRI site/CMV
enhancer/U6 promoter/XbaI site, followed by treating the produced
fragment treated with EcoRI and XbaI and then cloned into
pUC119.
[0056] The above plasmid prepared and treated with XbaI and HindIII
was joined to a fragment containing a tRNA.sup.Py1-terminator
isolated from the previously prepared tRNA.sup.VAL-tRNA.sup.Py1
tandem expression plasmid by XbaI and HindIII digestion, as a
result of which there was obtained an expression plasmid having a
DNA fragment of a nucleotide sequence set forth in SEQ ID NO:6. As
control, there was constructed a plasmid made up of pcDNA3.1+Zeo to
the multiple cloning site of which three tandem tRNA.sup.Py1 were
linked.
[0057] Likewise, using Escherichia coli suppressor tyrosine tRNA
instead of Methanosarcina mazei-originating wild type pyrrolysine
tRNA.sup.Py1, there was constructed a DNA fragment (SEQ ID NO:7)
that CMV enhancer and promoter of human U6 snRNA were linked to, to
produce an expression plasmid in the similar manner mentioned
above. Here, it has been reported that CMV enhancer activates RNA
transcription from U6 promoter.
(Construction of Reporter Gene Expression Plasmid)
[0058] Using Quick Change site-directed mutagenesis kit
(Stratagene), the leucine codon at position 111 of human grb2 was
converted to an amber codon (grb2 (111 amber)). Subsequently, a
gene (SEQ ID NO:12) constructed such that FLAG tag (DYKDDDDK) was
added to the C terminus thereof was incorporated into the
BamHI-XhoI site of pcDNA4/TO to produce a plasmid for detection of
suppression.
[0059] Likewise, a tyrosine codon at position 91 of Escherichia
coli's .beta.-galactosidase (lacZ) was converted to an amber codon
and cloned (lacZ (91 amber)) into a multiple cloning site of
pcDNA3.1+ (Zeo resistant).
(Introduction of Gene into Cell and Suppression Reaction)
Example 1
Grb2 Amber Suppression by tRNA.sup.Py1 Linked to Human Valine tRNA
Gene Promoter
[0060] Chinese hamster ovary cells cultivated in a 2.0 ml culture
scale 6-well plate (CHO cells; as subculture medium, DMEM/F-12
(Gibco), 10% FBS (ICN), 1/100 penicillin-streptomycin (Gibco) were
used) were provided with 0.5 .mu.g/well of three (different) types
of expression plasmids: Py1RS, tRNA.sup.Py1 linked to human valine
tRNA gene promoter, and grb2 (111 amber) in various combinations
thereof (see RESULT), and transfection was conducted under 90%
confluent state. The transfection was performed according to a
method using Lipofectamine 2000 (Invitrogen) and the manual (or
instruction) for the method (from Invitrogen). On the transfection,
Opti-MEM (Gibco) was used as culture medium. The transfected cell
culture medium (solution) was replaced with DMEM/F-12 (Gibco) in
the presence or absence of 1 mM N.epsilon.-Boc-lysine (Bachem),
induced expression was caused by addition of 1 .mu.g/mL
tetracycline, and incubation was conducted at 37 degrees Celsius
for ca. 20 hours in a CO.sub.2 incubator.
[0061] The above cultured cells from which the cell culture medium
(solution) was removed were washed with buffer solution, followed
by lysis of the cells to recover proteins. SDS-polyacrylamide gel
electrophoresis was performed to separate the proteins from each
other according to molecular weights thereof, followed by
electroblotting (100 V, 1 hour) to membrane. Regarding antibodies,
anti-FLAG M2 (Sigma) was used as primary antibody, and
sheep-originating whole antibody conjugated with horseradish
peroxidase for anti-mouse IgG (Amersham) was used as secondary
antibody. As a detection reagent, ECL western blotting detection
reagent (Amersham) was used. Measurement was conducted using a
cooled CCD camera LAS 1000 plus (Fuji Film).
[0062] FIG. 2 shows a result detected by western blotting of
suppression of Grb2 (111amb). Lane 1 on the left is a control for
showing the position of the band of full-length Grb2. Wild type
Grb2 has FLAG tag added to the C terminus thereof, and the band
thereof is detected at the position indicated by the arrow for Grb2
in cases where it is synthesized to the C terminus thereof. Lanes 2
to 4 show results in the case of lack of any one of Py1RS,
pyrrolysine tRNA and N.epsilon.-Boc-lysine. In these cases, a
full-length Grb2 was not synthesized. Contrary, lane 5 shows a
result in the case of all of Py1RS, pyrrolysine tRNA and
N.epsilon.-Boc-lysine being introduced into a cell, wherein the
full-length Grb2 was synthesized. It is apparent from the results
that by Py1RS and pyrrolysine tRNA, N.epsilon.-Boc-lysine was
incorporated into an amber codon incorporated into position 111 of
Grb2.
Example 2
Grb2 Amber Suppression by tRNA.sup.Tyr and tRNA.sup.Py1 Linked to
U6 Promoter
[0063] Subsequently, human Grb2 gene, wild type TyrRS, and Py1RS,
which were prepared using a similar method in the above Example,
were expressed and were subjected to suppression by Methanosarcina
mazei-originating wild type pyrrolysine tRNA.sup.Py1 having U6
promoter or Escherichia coli suppressor tRNA.sup.Tyr. FIG. 3 shows
a result detected by western blotting of suppression of Grb2
(111amb). Lane on the most right is a control for showing the
position of bands of wild type Grb2. Two lanes on the left show
results in cases where Escherichia coli suppressor tRNA.sup.Tyr
having U6 promoter was expressed. From the band of Grb2 having been
detected depending on expression of TryRS, it is apparent that
tyrosine was incorporated into the amber codon of Grb2. Two lanes
in the middle show results in cases where pyrrolysine tRNA.sup.Py1
having U6 promoter was expressed. From the band of Grb2 having been
detected by addition of N.epsilon.-Boc-lysine to culture (medium),
it is apparent that N.epsilon.-Boc-lysine was incorporated into the
amber codon of Grb2. The results revealed that tRNA gene having U6
promoter linked to its 5' end was transcribed in a mammalian cell.
Control experiment in which tyrosine is not added to medium in the
case of tyrosine tRNA being expressed was not conducted because
cells do not grow in the absence of tyrosine.
Example 3
lacZ Amber Suppression by tRNA.sup.Py1
[0064] Chinese hamster ovary cells (CHO-TRex cells) were given to
24-well plate by 1.2.times.10.sup.5 cells/well and incubated in
DMEM/F-12 culture media (Gibco) containing 10% fetal bovine serum
(ICN) and 1/100 penicillin-streptomycin (Gibco). Next day, when the
media was 95% confluent, transfection was conducted by using 0.4
.mu.g lacZ (91 amber), 0.2 .mu.g Py1RS expression plasmids, and
three different suppressor tRNA.sup.Py1 expression plasmids (each
containing human valine tRNA, U6 promoter, and CMV enhancer)
prepared in the above Example. The transfection was conducted using
2 .mu.l Lipofectamine 2000 (Invitrogen) according to the manual
(instruction) thereof. On the transfection, Opti-MEM (Gibco) was
used as culture medium.
[0065] The transducted cell culture medium was replaced with
DMEM/F-12 (Gibco) in the presence or absence of 1 mM Boc-lysine
(Bachem), induced expression was caused by addition of 1 .mu.g/mL
tetracycline, and incubation was conducted at 37 degrees Celsius
for ca. 20 hours in a CO.sub.2 incubator. Next day, proteins were
recovered from the cells, and lacZ enzyme activities thereof were
examined using a reporter assay kit .beta.-Gal (TOYOBO). The result
is shown in FIG. 4. It is apparent therefrom that in either case
where human valine tRNA promoter or U6 promoter was used,
.beta.-galactosidase activity was detected by addition of
Boc-lysine to medium, and thus the amber codon of lacZ gene was
subjected to suppression. On the contrary, in the case of tRNA
expression vector using CMV promoter not containing the above
promoters, suppression was not caused because .beta.-galactosidase
activity was not detected regardless of whether Boc-lysine was
added or not. It is assumed that expression of suppressor tRNA by
U6 promoter is significantly high as compared with that by human
valine tRNA promoter.
Example 4
lacZ Amber Suppression by tRNA.sup.Tyr Linked to U6 Promoter
[0066] By a method similar to that of Example 3, U6 promoter-linked
Escherichia coli suppressor tRNA.sup.Tyr gene and three different
mutant TyrRS expression plasmids were expressed, and lacZ amber
suppression was performed by addition of three types of tyrosine
derivatives of iodotyrosine (IY), azidophenylalanine (AzPhe), and
parabenzoylphenylalanine (pBpa). The result is shown in FIG. 5. It
is apparent therefrom that in each case where any one of the amino
acids was added, significantly high .beta.-galactosidase activity
was detected as compared with the case where no amino acids were
added, and thus the amber codon of lacZ gene was suppressed. In
this regard, it seems that the detection of .beta.-galactosidase
activity even in the absence of iodotyrosine is due to the
suppression caused even in the case of no addition of IY by
IY-specific mutant TyrRS, which incorporates not only iodotyrosine
but also tyrosine.
Reference Example 1
[0067] FIG. 6 shows data of mass spectrometry indicating that in
Escherichia coli, N.epsilon.-Boc-lysine was incorporated into a
peptide in the presence of Py1RS and pyrrolysine tRNA. As shown in
FIG. 6, the peak of molecular weight (MW) 1327.67 indicates a
peptide whose sequence is NSYSPILGYWK. The peak of molecular weight
1392.76 indicates a peptide whose tyrosine presented at 11.sup.th
(sic, 9th) position from the left was replaced with
N.epsilon.-Boc-lysine (which is indicated with the mark "*").
Example 5
Construction of Expression System of Suppressor tRNA by T7
Promoter, and lacZ Amber Suppression
[0068] T7 RNA polymerase gene was amplified by PCR and cloned into
between EcoRI and XhoI of pcDNA4/TO to produce T7 RNA polymerase
expression plasmid. In order to produce T7-tRNA.sup.Tyr gene,
first, there was conducted PCR by using U6-tRNA.sup.Tyr (SEQ ID
NO:7) as template, thereby adding T7 promoter to tRNA.sup.Tyr
sequence, to be cloned into pCR4blunt-TOPO. Then, T7-tRNA.sup.Tyr
gene was cut out by treatment of EcoRI, to be cloned into EcoRI
site of pBR322. The so prepared T7-tRNA.sup.Tyr gene and a portion
of (so prepared) plasmid sequence (SEQ ID NO:15) were amplified by
PCR, and DNA obtained was purified and used to transform cells. In
order to construct tRNA.sup.Py1 expression plasmid, the pBR322 into
which T7-tRNA.sup.Tyr gene was cloned as mentioned above was
treated with XbaI and HindIII, followed by isolation of a fragment
containing tRNA.sup.Py1-terminator from tRNA expression plasmid
with U6 promoter by means of XbaI and HindIII digestions, to couple
them with each other. The so prepared T7-tRNA.sup.Py1 gene and a
portion of (so prepared) plasmid sequence (SEQ ID NO:14) were
amplified by PCR, and DNA obtained was purified and used to
transform cells.
[0069] Using a method similar to that of Example 3, transfection
was performed by using 0.2 .mu.g T7-tRNA.sup.Py1 expression
plasmid, 0.1 .mu.g Py1RS expression plasmid, 0.4 .mu.g lacZ (91
amber) expression plasmid, and 0.3 .mu.g T7 RNA polymerase
expression plasmid to conduct lacZ amber suppression, except that
incubation was conducted in DMEM/F-12 culture medium (Gibco)
without 1/100 penicillin-streptomycin (Gibco). The result is shown
in FIG. 7. It is apparent therefrom that in the case where T7 RNA
polymerase was expressed (T7RNAP+), significantly high
.beta.-galactosidase activity was detected as compared with the
case where T7 RNA polymerase was not expressed (T7RNAP-), and thus
the amber codon of lacZ gene was suppressed.
[0070] Using a method similar to that of Example 3, transfection
was performed by using 0.18 .mu.g T7-tRNA.sup.Tyr gene DNA, 0.1
.mu.g TyrRS expression plasmid, 0.4 .mu.g lacZ (91 amber)
expression plasmid, and 0.3 .mu.g T7 RNA polymerase expression
plasmid to conduct lacZ amber suppression. However, incubation was
conducted in DMEM/F-12 culture medium (Gibco) without 1/100
penicillin-streptomycin (Gibco). The result is shown in FIG. 8. It
is apparent therefrom that in the case where T7 RNA polymerase was
expressed, significantly high .beta.-galactosidase activity was
detected as compared with the case where T7 RNA polymerase was not
expressed, and thus the amber codon of lacZ gene was
suppressed.
Example 6
Construction of Expression System of Suppressor tRNA by
U1snRNA-Type Transcription Promoter, and lacZ Amber Suppression
[0071] Construction of tRNA expression plasmid by U1snRNA
type-promoter was conducted according to the following method.
Using the previously prepared U6-tRNA Try [sic] (SEQ ID NO:7) as
template, the region from 198 bases upstream of U6 promoter
transcription initiation site to upstream of TATA box was amplified
by using the following primers:
TABLE-US-00001 5'-ATGATATCAGAGGGCCTATTTCCCAT-3' (SEQ ID NO:16)
5'-TGCTCGAGAAGCCAAGAATCGAAATAC-3'. (SEQ ID NO:17)
[0072] This region includes transcription element PSE, wherein the
amplified DNA fragment has EcoRV site added at its 5' end and XhoI
site added at its 3' end. The PCR product was once integrated into
EcoRV-XhoI site of plasmid pcDNA3.1+. Vector-originating EcoO109I
and NotI sites are present downstream of XhoI site. A sequence
downstream of TATA box of U6 promoter and a terminator for stopping
transcription by polymerase III were inserted into between these
XhoI and EcoO109I. After insertion of tRNA.sup.Tyr sequence into
EcoO109I site, 3' box, which is a terminator of polymerase II, was
further inserted into NotI site. The whole region from EcoRV
through 3' box constitutes PSE-tRNA.sup.Tyr gene (SEQ ID NO:18).
This gene was amplified by PCR and cloned into pCR4blunt-TOPO to
produce a plasmid, which corresponds to PSE-tRNA.sup.Tyr expression
plasmid. The so prepared PSE-tRNA.sup.Tyr gene has U6 promoter from
which TATA sequence is removed so that transcription by RNA
polymerase II is caused (Das et al., Nature 1995, Vol. 374, pp.
657-660). In addition, PCR amplification was performed using the
following two primers having CMV enhancers similar to those of U6
promoter:
TABLE-US-00002 (SEQ ID NO:19)
5'-ATCGAATTCTAGTTATTAATAGTAATCAATTACG-3' and (SEQ ID NO:20)
5'-AGCCTTGTATCGTATATGC-3',
and 5' phosphorylation was further conducted, followed by insertion
into EcoRI site of PSE-tRNA.sup.Tyr gene to produce
CMV-DSE-PSE-tRNA.sup.Tyr.
[0073] Using a method similar to that of Example 3, transfection
was conducted by using 0.2 .mu.g of PSE-tRNA.sup.Tyr expression
plasmid or CMV-PSE-tRNA.sup.Tyr expression plasmid, 0.2 .mu.g of
TyrRS expression plasmid, and 0.4 .mu.g of lacZ (91 amber)
expression plasmid. On the day following the transfection, cells
were stained using .beta.-Galactosidase Staining Kit (Mirus) to
examine whether amber suppression of lacZ was caused. A cell having
caused suppression is expected to be stained blue. FIG. 9 shows
photographs depicting results of staining cells, wherein stained
cells are indicated with arrows. Presence or absence of the
enhancer that has not caused particularly large difference, the
suppression activities, even though low, were confirmed in both
cases.
INDUSTRIAL APPLICABILITY
[0074] The present invention is able to effectively produce
alloprotein(s) into which there is incorporated non-natural amino
acid such as lysine derivative, tyrosine derivative etc.
Sequence CWU 1
1
20112DNAArtificial Sequencemisc_signalconsensus sequence of Type II
promoter 1trgcnnagyn gg 12211DNAArtificial
Sequencemisc_signalconsensus sequence of Type II promoter
2ggttcgantc c 113250DNAArtificial SequenceHuman U6 snRNA promoter
3agagggccta tttcccatga ttccttcata tttgcatata cgatacaagg ctgttagaga
60gataattaga attaatttga ctgtaaacac aaagatatta gtacaaaata cgtgacgtag
120aaagtaataa tttcttgggt agtttgcagt tttaaaatta tgttttaaaa
tggactatca 180tatgcttacc gtaacttgaa agtatttcga tttcttggct
ttatatatct tgtggaaagg 240acgaaacacc 250472RNAMethanosarcina mazeii
4ggaaaccuga ucauguagau cgaauggacu cuaaauccgu ucagccgggu uagauucccg
60ggguuuccgc ca 72585RNAEscherichia coli 5ggugggguuc ccgagcggcc
aaagggagca gacucuaaau cugccgucau cgacuucgaa 60gguucgaauc cuucccccac
cacca 856852DNAArtificial SequenceU6-tRNAPyl expression construct
6tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg
60cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
120gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
attgacgtca 180atgggtggag tatttacggt aaactgccca cttggcagta
catcaagtgt atcatatgcc 240aagtacgccc cctattgacg tcaatgacgg
taaatggccc gcctggcatt atgcccagta 300catgacctta tgggactttc
ctacttggca gtacatctac gtattagtca tcgctattac 360catggtgatg
cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
420atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
aaaatcaacg 480ggacagaggg cctatttccc atgattcctt catatttgca
tatacgatac aaggctgtta 540gagagataat tagaattaat ttgactgtaa
acacaaagat attagtacaa aatacgtgac 600gtagaaagta ataatttctt
gggtagtttg cagttttaaa attatgtttt aaaatggact 660atcatatgct
taccgtaact tgaaagtatt tcgatttctt ggctttatat atcttgtgga
720aaggacgaaa caccgagatc ttctagactc gagggaaacc tgatcatgta
gatcgaatgg 780actctaaatc cgttcagccg ggttagattc ccggggtttc
cggacaagtg cggttttttt 840ctccagctcc cg 8527865DNAArtificial
SequenceU6-tRNATyr expression construct 7tagttattaa tagtaatcaa
ttacggggtc attagttcat agcccatata tggagttccg 60cgttacataa cttacggtaa
atggcccgcc tggctgaccg cccaacgacc cccgcccatt 120gacgtcaata
atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
180atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
atcatatgcc 240aagtacgccc cctattgacg tcaatgacgg taaatggccc
gcctggcatt atgcccagta 300catgacctta tgggactttc ctacttggca
gtacatctac gtattagtca tcgctattac 360catggtgatg cggttttggc
agtacatcaa tgggcgtgga tagcggtttg actcacgggg 420atttccaagt
ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
480ggacagaggg cctatttccc atgattcctt catatttgca tatacgatac
aaggctgtta 540gagagataat tagaattaat ttgactgtaa acacaaagat
attagtacaa aatacgtgac 600gtagaaagta ataatttctt gggtagtttg
cagttttaaa attatgtttt aaaatggact 660atcatatgct taccgtaact
tgaaagtatt tcgatttctt ggctttatat atcttgtgga 720aaggacgaaa
caccgagatc ttctagactc gagggtgggg ttcccgagcg gccaaaggga
780gcagactcta aatctgccgt catcgacttc gaaggttcga atccttcccc
caccagacaa 840gtgcggtttt tttctccagc tcccg 86581392DNAMethanosarcina
mazeiiCDS(1)..(1392) 8atg gac tac aag gac gac gat gac aag atg gat
aaa aaa cca cta aac 48Met Asp Tyr Lys Asp Asp Asp Asp Lys Met Asp
Lys Lys Pro Leu Asn1 5 10 15act ctg ata tct gca acc ggg ctc tgg atg
tcc agg acc gga aca att 96Thr Leu Ile Ser Ala Thr Gly Leu Trp Met
Ser Arg Thr Gly Thr Ile20 25 30cat aaa ata aaa cac cac gaa gtc tct
cga agc aaa atc tat att gaa 144His Lys Ile Lys His His Glu Val Ser
Arg Ser Lys Ile Tyr Ile Glu35 40 45atg gca tgc gga gac cac ctt gtt
gta aac aac tcc agg agc agc agg 192Met Ala Cys Gly Asp His Leu Val
Val Asn Asn Ser Arg Ser Ser Arg50 55 60act gca aga gcg ctc agg cac
cac aaa tac agg aag acc tgc aaa cgc 240Thr Ala Arg Ala Leu Arg His
His Lys Tyr Arg Lys Thr Cys Lys Arg65 70 75 80tgc agg gtt tcg gat
gag gat ctc aat aag ttc ctc aca aag gca aac 288Cys Arg Val Ser Asp
Glu Asp Leu Asn Lys Phe Leu Thr Lys Ala Asn85 90 95gaa gac cag aca
agc gta aaa gtc aag gtc gtt tct gcc cct acc aga 336Glu Asp Gln Thr
Ser Val Lys Val Lys Val Val Ser Ala Pro Thr Arg100 105 110acg aaa
aag gca atg cca aaa tcc gtt gcg aga gcc ccg aaa cct ctt 384Thr Lys
Lys Ala Met Pro Lys Ser Val Ala Arg Ala Pro Lys Pro Leu115 120
125gag aat aca gaa gcg gca cag gct caa cct tct gga tct aaa ttt tca
432Glu Asn Thr Glu Ala Ala Gln Ala Gln Pro Ser Gly Ser Lys Phe
Ser130 135 140cct gcg ata ccg gtt tcc acc caa gag tca gtt tct gtc
ccg gca tct 480Pro Ala Ile Pro Val Ser Thr Gln Glu Ser Val Ser Val
Pro Ala Ser145 150 155 160gtt tca aca tca ata tca agc att tct aca
gga gca act gca tcc gca 528Val Ser Thr Ser Ile Ser Ser Ile Ser Thr
Gly Ala Thr Ala Ser Ala165 170 175ctg gta aaa ggg aat acg aac ccc
att aca tcc atg tct gcc cct gtt 576Leu Val Lys Gly Asn Thr Asn Pro
Ile Thr Ser Met Ser Ala Pro Val180 185 190cag gca agt gcc ccc gca
ctt acg aag agc cag act gac agg ctt gaa 624Gln Ala Ser Ala Pro Ala
Leu Thr Lys Ser Gln Thr Asp Arg Leu Glu195 200 205gtc ctg tta aac
cca aaa gat gag att tcc ctg aat tcc ggc aag cct 672Val Leu Leu Asn
Pro Lys Asp Glu Ile Ser Leu Asn Ser Gly Lys Pro210 215 220ttc agg
gag ctt gag tcc gaa ttg ctc tct cgc aga aaa aaa gac ctg 720Phe Arg
Glu Leu Glu Ser Glu Leu Leu Ser Arg Arg Lys Lys Asp Leu225 230 235
240cag cag atc tac gcg gaa gaa agg gag aat tat ctg ggg aaa ctc gag
768Gln Gln Ile Tyr Ala Glu Glu Arg Glu Asn Tyr Leu Gly Lys Leu
Glu245 250 255cgt gaa att acc agg ttc ttt gtg gac agg ggt ttt ctg
gaa ata aaa 816Arg Glu Ile Thr Arg Phe Phe Val Asp Arg Gly Phe Leu
Glu Ile Lys260 265 270tcc ccg atc ctg atc cct ctt gag tat atc gaa
agg atg ggc att gat 864Ser Pro Ile Leu Ile Pro Leu Glu Tyr Ile Glu
Arg Met Gly Ile Asp275 280 285aat gat acc gaa ctt tca aaa cag atc
ttc agg gtt gac aag aac ttc 912Asn Asp Thr Glu Leu Ser Lys Gln Ile
Phe Arg Val Asp Lys Asn Phe290 295 300tgc ctg aga ccc atg ctt gct
cca aac ctt tac aac tac ctg cgc aag 960Cys Leu Arg Pro Met Leu Ala
Pro Asn Leu Tyr Asn Tyr Leu Arg Lys305 310 315 320ctt gac agg gcc
ctg cct gat cca ata aaa att ttt gaa ata ggc cca 1008Leu Asp Arg Ala
Leu Pro Asp Pro Ile Lys Ile Phe Glu Ile Gly Pro325 330 335tgc tac
aga aaa gag tcc gac ggc aaa gaa cac ctc gaa gag ttt acc 1056Cys Tyr
Arg Lys Glu Ser Asp Gly Lys Glu His Leu Glu Glu Phe Thr340 345
350atg ctg aac ttc tgc cag atg gga tcg gga tgc aca cgg gaa aat ctt
1104Met Leu Asn Phe Cys Gln Met Gly Ser Gly Cys Thr Arg Glu Asn
Leu355 360 365gaa agc ata att aca gac ttc ctg aac cac ctg gga att
gat ttc aag 1152Glu Ser Ile Ile Thr Asp Phe Leu Asn His Leu Gly Ile
Asp Phe Lys370 375 380atc gta ggc gat tcc tgc atg gtc tat ggg gat
acc ctt gat gta atg 1200Ile Val Gly Asp Ser Cys Met Val Tyr Gly Asp
Thr Leu Asp Val Met385 390 395 400cac gga gac ctg gaa ctt tcc tct
gca gta gtc gga ccc ata ccg ctt 1248His Gly Asp Leu Glu Leu Ser Ser
Ala Val Val Gly Pro Ile Pro Leu405 410 415gac cgg gaa tgg ggt att
gat aaa ccc tgg ata ggg gca ggt ttc ggg 1296Asp Arg Glu Trp Gly Ile
Asp Lys Pro Trp Ile Gly Ala Gly Phe Gly420 425 430ctc gaa cgc ctt
ctc aag gtt aaa cac gac ttt aaa aat atc aag aga 1344Leu Glu Arg Leu
Leu Lys Val Lys His Asp Phe Lys Asn Ile Lys Arg435 440 445gct gca
agg tcc ggg tct tac tat aac ggg att tct acc aac ctg taa 1392Ala Ala
Arg Ser Gly Ser Tyr Tyr Asn Gly Ile Ser Thr Asn Leu450 455
4609463PRTMethanosarcina mazeii 9Met Asp Tyr Lys Asp Asp Asp Asp
Lys Met Asp Lys Lys Pro Leu Asn1 5 10 15Thr Leu Ile Ser Ala Thr Gly
Leu Trp Met Ser Arg Thr Gly Thr Ile20 25 30His Lys Ile Lys His His
Glu Val Ser Arg Ser Lys Ile Tyr Ile Glu35 40 45Met Ala Cys Gly Asp
His Leu Val Val Asn Asn Ser Arg Ser Ser Arg50 55 60Thr Ala Arg Ala
Leu Arg His His Lys Tyr Arg Lys Thr Cys Lys Arg65 70 75 80Cys Arg
Val Ser Asp Glu Asp Leu Asn Lys Phe Leu Thr Lys Ala Asn85 90 95Glu
Asp Gln Thr Ser Val Lys Val Lys Val Val Ser Ala Pro Thr Arg100 105
110Thr Lys Lys Ala Met Pro Lys Ser Val Ala Arg Ala Pro Lys Pro
Leu115 120 125Glu Asn Thr Glu Ala Ala Gln Ala Gln Pro Ser Gly Ser
Lys Phe Ser130 135 140Pro Ala Ile Pro Val Ser Thr Gln Glu Ser Val
Ser Val Pro Ala Ser145 150 155 160Val Ser Thr Ser Ile Ser Ser Ile
Ser Thr Gly Ala Thr Ala Ser Ala165 170 175Leu Val Lys Gly Asn Thr
Asn Pro Ile Thr Ser Met Ser Ala Pro Val180 185 190Gln Ala Ser Ala
Pro Ala Leu Thr Lys Ser Gln Thr Asp Arg Leu Glu195 200 205Val Leu
Leu Asn Pro Lys Asp Glu Ile Ser Leu Asn Ser Gly Lys Pro210 215
220Phe Arg Glu Leu Glu Ser Glu Leu Leu Ser Arg Arg Lys Lys Asp
Leu225 230 235 240Gln Gln Ile Tyr Ala Glu Glu Arg Glu Asn Tyr Leu
Gly Lys Leu Glu245 250 255Arg Glu Ile Thr Arg Phe Phe Val Asp Arg
Gly Phe Leu Glu Ile Lys260 265 270Ser Pro Ile Leu Ile Pro Leu Glu
Tyr Ile Glu Arg Met Gly Ile Asp275 280 285Asn Asp Thr Glu Leu Ser
Lys Gln Ile Phe Arg Val Asp Lys Asn Phe290 295 300Cys Leu Arg Pro
Met Leu Ala Pro Asn Leu Tyr Asn Tyr Leu Arg Lys305 310 315 320Leu
Asp Arg Ala Leu Pro Asp Pro Ile Lys Ile Phe Glu Ile Gly Pro325 330
335Cys Tyr Arg Lys Glu Ser Asp Gly Lys Glu His Leu Glu Glu Phe
Thr340 345 350Met Leu Asn Phe Cys Gln Met Gly Ser Gly Cys Thr Arg
Glu Asn Leu355 360 365Glu Ser Ile Ile Thr Asp Phe Leu Asn His Leu
Gly Ile Asp Phe Lys370 375 380Ile Val Gly Asp Ser Cys Met Val Tyr
Gly Asp Thr Leu Asp Val Met385 390 395 400His Gly Asp Leu Glu Leu
Ser Ser Ala Val Val Gly Pro Ile Pro Leu405 410 415Asp Arg Glu Trp
Gly Ile Asp Lys Pro Trp Ile Gly Ala Gly Phe Gly420 425 430Leu Glu
Arg Leu Leu Lys Val Lys His Asp Phe Lys Asn Ile Lys Arg435 440
445Ala Ala Arg Ser Gly Ser Tyr Tyr Asn Gly Ile Ser Thr Asn Leu450
455 4601018DNAArtificial SequenceLinker DNA 10agatcttcta gactcgag
1811245DNAArtificial SequencetRNAVal-tRNA pyl tandem expression
vector 11agcgctccgg tttttctgtg ctgaacctca ggggacgccg acacacgtac
acgtcgtttc 60cgtagtgtag tggtcatcac gttcgcctaa cacgcgaaag gtccccggtt
cgaaaccggg 120cggaaacaag atcttctaga ctcgagggaa acctgatcat
gtagatcgaa tggactctaa 180atccgttcag ccgggttaga ttcccggggt
ttccggacaa gtgcggtttt tttctccagc 240tcccg 24512678DNAArtificial
SequenceHuman Grb2 suppresion reporter gene 12atg gaa gcc atc gcc
aaa tat gac ttc aaa gct act gca gac gac gag 48Met Glu Ala Ile Ala
Lys Tyr Asp Phe Lys Ala Thr Ala Asp Asp Glu1 5 10 15ctg agc ttc aaa
agg ggg gac atc ctc aag gtt ttg aac gaa gaa tgt 96Leu Ser Phe Lys
Arg Gly Asp Ile Leu Lys Val Leu Asn Glu Glu Cys20 25 30gat cag aac
tgg tac aag gca gag ctt aat gga aaa gac ggc ttc att 144Asp Gln Asn
Trp Tyr Lys Ala Glu Leu Asn Gly Lys Asp Gly Phe Ile35 40 45ccc aag
aac tac ata gaa atg aaa cca cat ccg tgg ttt ttt ggc aaa 192Pro Lys
Asn Tyr Ile Glu Met Lys Pro His Pro Trp Phe Phe Gly Lys50 55 60atc
ccc aga gcc aag gca gaa gaa atg ctt agc aaa cag cgg cac gat 240Ile
Pro Arg Ala Lys Ala Glu Glu Met Leu Ser Lys Gln Arg His Asp65 70 75
80ggg gcc ttt ctt atc cga gag agt gag agc gct cct ggg gac ttc tcc
288Gly Ala Phe Leu Ile Arg Glu Ser Glu Ser Ala Pro Gly Asp Phe
Ser85 90 95ctc tct gtc aag ttt gga aac gat gtg cag cac ttc aag gtg
tag cga 336Leu Ser Val Lys Phe Gly Asn Asp Val Gln His Phe Lys Val
Arg100 105 110gat gga gcc ggg aag tac ttc ctc tgg gtg gtg aag ttc
aat tct ttg 384Asp Gly Ala Gly Lys Tyr Phe Leu Trp Val Val Lys Phe
Asn Ser Leu115 120 125aat gag ctg gtg gat tat cac aga tct aca tct
gtc tcc aga aac cag 432Asn Glu Leu Val Asp Tyr His Arg Ser Thr Ser
Val Ser Arg Asn Gln130 135 140cag ata ttc ctg cgg gac ata gaa cag
gtg cca cag cag ccg aca tac 480Gln Ile Phe Leu Arg Asp Ile Glu Gln
Val Pro Gln Gln Pro Thr Tyr145 150 155gtc cag gcc ctc ttt gac ttt
gat ccc cag gag gat gga gag ctg ggc 528Val Gln Ala Leu Phe Asp Phe
Asp Pro Gln Glu Asp Gly Glu Leu Gly160 165 170 175ttc cgc cgg gga
gat ttt atc cat gtc atg gat aac tca gac ccc aac 576Phe Arg Arg Gly
Asp Phe Ile His Val Met Asp Asn Ser Asp Pro Asn180 185 190tgg tgg
aaa gga gct tgc cac ggg cag acc ggc atg ttt ccc cgc aat 624Trp Trp
Lys Gly Ala Cys His Gly Gln Thr Gly Met Phe Pro Arg Asn195 200
205tat gtc acc ccc gtg aac cgg aac gtc gac tac aag gac gac gat gac
672Tyr Val Thr Pro Val Asn Arg Asn Val Asp Tyr Lys Asp Asp Asp
Asp210 215 220aag tga 678Lys1317DNAArtificial SequenceT7 promoter
13taatacgact cactata 1714248DNAArtificial SequenceT7-tRNAPyl
expression construct 14ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg
tctaagaaac cattattatc 60atgacattaa cctataaaaa taggcgtatc acgaggccct
ttcgtcttca agaattcgcc 120ctttaatacg actcactata gggagatctt
ctagactcga gggaaacctg atcatgtaga 180tcgaatggac tctaaatccg
ttcagccggg ttagattccc ggggtttccg gacaagtgcg 240gttttttt
24815263DNAArtificial SequenceT7-tRNATyr expression construct
15ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tctaagaaac cattattatc
60atgacattaa cctataaaaa taggcgtatc acgaggccct ttcgtcttca agaattcgcc
120ctttaatacg actcactata gggagatctt ctagactcga gggtggggtt
cccgagcggc 180caaagggagc agactctaaa tctgccgtca tcgacttcga
aggttcgaat ccttccccca 240ccagacaagt gcggtttttt ttt
2631626DNAArtificial SequenceForward primer 16atgatatcag agggcctatt
tcccat 261727DNAArtificial SequenceReverse primer 17tgctcgagaa
gccaagaatc gaaatac 2718408DNAArtificial SequencePSE-tRNATyr
expression construct 18gatatcagag agataattag aattaatttg actgtaaaca
caaagatatt agtacaaaat 60acgtgacgta gaaagtaata atttcttggg tagtttgcag
ttttaaaatt atgttttaaa 120atggactatc atatgcttac cgtaacttga
aagtatttcg atttcttggc ttctcgagcc 180ttgtggaaag gacgaaacac
cgcttaaggg cccgtttttc caagatcttc tagactcgag 240ggtggggttc
ccgagcggcc aaagggagca gactctaaat ctgccgtcat cgacttcgaa
300ggttcgaatc cttcccccac cagacaagtg cggttttttt ctccagctcc
cgaagcttgc 360ggccgctttt ttttggagtt tcaaaagtag acagcggccg cagggccc
4081934DNAArtificial SequenceCMV-primer1 19atcgaattct agttattaat
agtaatcaat tacg 342019DNAArtificial SequenceCMV-primer2
20agccttgtat cgtatatgc 19
* * * * *